Diagnóstico genético preimplantatorio: implantación, desarrollo y actualización de un programa de diagnóstico genético preimplantatorio en un Sistema Sanitario Público.

Falta palabras claveEl Diagnóstico Genético Preimplantatorio (PGD) surge con el objetivo de ofrecer una opción reproductiva a familias con alto riesgo de transmitir enfermedades de base genética a su descendencia. Básicamente consiste en analizar genéticamente los preembriones (terminología legal para definir embriones de menos de 14 días de desarrollo) obtenidos por técnicas de fecundación in vitro, de forma que sólo son transferidos al útero los no afectos de la enfermedad de riesgo. El análisis genético se realiza sobre una o dos células extraídas al preembrión cuando éste tiene 3 días de desarrollo. El resultado será representativo del resto del embrión. Podría decirse que es una forma precoz de Diagnóstico Prenatal (DP), pero con una ventaja principal respecto a este, como es evitar la interrupción voluntaria del embarazo por enfermedad fetal, pues los progenitores no tienen que pasar por el trance de decidir si continúan o no con una gestación ya establecida afecta de una enfermedad grave que ellos portan o padecen. El PGD contribuye a la interrupción de la transmisión familiar de las enfermedades hereditarias, que supone un alivio del sufrimiento familiar, y tiene gran importancia social y sanitaria al reducir el número de pacientes afectos de estas dolencias.En España, el PGD está contemplado en la actual Ley de Reproducción Humana Asistida (Ley 14/2006 de 26 de mayo, Artículo 12), donde se especifica cuáles son las indicaciones susceptibles de PGD permitidas en nuestro país y los pasos administrativos para poder realizarlo. Andalucía concretamente, ha sido la primera Comunidad donde se ha incorporado y regulado la realización del PGD en el ámbito público, mediante el Decreto 156/2005, de 28 de junio, “… por el que se regula el PGD en el Sistema Sanitario Público de Andalucía (SSPA), se crea la Comisión Andaluza de Genética y Reproducción y se designa a la Unidad de Gestión Clínica de Genética, Reproducción y Medicina Fetal (UGCGRMF) como centro de referencia dentro del SSPA”. (Esta Unidad hoy día es conocida como Unidad de Gestión Clínica de Medicina Materno-fetal, Genética y Reproducción, de Hospital Virgen de Rocío-Virgen Macarena). Dicho Decreto contempla el Anexo II, donde se recoge un listado de enfermedades inicialmente incluidas en el programa de PGD del SSPA, que ha sido posteriormente ampliado. El objeto fundamental de esta tesis doctoral ha sido proporcionar al programa de PGD de la UGCGRMF de nuevas estrategias diagnósticas, con el fin de llegar a un mayor número de familias afectadas por Enfermedades Raras. Todo ello a lo largo de 10 años, desde su implantación hasta la actualidad. Para ello se han optimizado los protocolos basados en PCR de todas las enfermedades incluidas en el Decreto156/2005 y en su posterior ampliación, se ha optimizado e incorporado la metodología MDA (Multiple Displacement Amplification) para el diagnóstico en un determinado grupo de familias y se ha iniciado la valoración para una posterior incorporación de la CGH (Hibridación Genómica Comparada) al programa, con el objetivo de diagnosticar una enfermedad monogénica y detectar aneuplodías y otros reordenamientos cromosómicos desequilibrados en una única célula. Todos los resultados del balance del programa son presentados

Experience of Preimplantation Genetic Diagnosis with HLA Matching at the University Hospital Virgen del Rocío in Spain: Technical and Clinical Overview

Preimplantation genetic diagnosis (PGD) of genetic diseases, combined with HLA matching (PGD-HLA), is an option for couples at risk of transmitting a genetic disease to select unaffected embryos of an HLA tissue type compatible with that of an existing affected child. Here we present the results of our PGD-HLA program at the Department of Genetics, Reproduction and Fetal Medicine of the University Hospital Virgen del Roc´ıo in Seville. Seven couples have participated in our program because of different indications. Overall, 26 cycles were performed, providing a total of 202 embryos. A conclusive molecular diagnosis and HLA-typing could be assured in 96% of the embryos. The percentage of transfers per cycle was 26.9% and the birth rate per cycle was 7.7% per transfer. Our PGD-HLA program resulted in the birth of 2 healthy babies, HLA-identical to their affected siblings, with successful subsequent haematopoietic stem cell (HSC) transplantations. Both HSC-transplanted children are currently doing well 48 and 21 months following transplantation, respectively. All the procedures, including HSCs umbilical cord transplantation, were performed in our hospital

Derivation of HVR1, HVR2 and HVR3 human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis (PGD) for monogenic disorder

From 106 human blastocyts donate for research after in vitro fertilization (IVF) and preimplantation genetic diagnosis (PGD) for monogenetic disorder, 3 human embryonic stem cells (hESCs) HVR1, HVR2 and HVR3 were successfully derived. HVR1 was assumed to be genetically normal, HVR2 carrying Becker muscular dystrophy and HVR3 Hemophilia B. Despite the translocation t(9;15)(q34.3;q14) detected in HVR2, all the 3 cell lines were characterised in vitro and in vivo as normal hESCs lines and were registered in the Spanish Stem Cell Bank.Junta de Andalucía FEDER TCMR0021/06, PI246-2008Instituto de Salud Carlos III (FEDER) RD12/0019/0028, RD012/0036/0017, PI10/00964, PI11/02923, PI14/0101

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Novel One-Step Multiplex PCR-Based Method for HLA Typing and Preimplantational Genetic Diagnosis of B-Thalassemia

Preimplantation genetic diagnosis (PGD) of single gene disorders, combined with HLA matching (PGD-HLA), has emerged as a tool for couples at risk of transmitting a genetic disease to select unaffected embryos of an HLA tissue type compatible with that of an existing affected child. Here, we present a novel one-step multiplex PCR to genotype a spectrum of STRs to simultaneously perform HLA typing and PGD for B-thalassemia. This method is being routinely used for PGD-HLA cycles in our department, with a genotyping success rate of 100%. As an example, we present the first successful PGD-HLA typing in Spain, which resulted in the birth of a boy and subsequent successful HSC transplantation to his affected brother, who is doing well 4 years following transplantation. The advantage of our method is that it involves only a round of single PCR for multiple markers amplification (up to 10 markers within the HLA and 6 markers at the B-globin loci). This strategy has allowed us to considerably reduce the optimization of the PCR method for each specific PGD-HLA family as well as the time to obtain molecular results in each cycle

Preimplantation Genetic Diagnosis for Myotonic Dystrophy Type 1 and Analysis of the Effect of the Disease on the Reproductive Outcome of the Affected Female Patients

Myotonic dystrophy type 1 (DM1) is the most common adult muscular dystrophy and presents an autosomal dominant inheritance. A reproductive option for the families affected is preimplantation genetic diagnosis (PGD). One limitation of this option is the nonoptimal response to ovarian stimulation of the women with DM1, although controversial results exist regarding this subject. In this study, we have analyzed the results of the PGD program applied to DM1 at our institution. A total of 35 couples have been included in our program since 2010, and 59 cycles have been performed. The percentage of transfers per cycle was 64.4% and the live birth rate per cycle was 18.6%. Interestingly, statistically significant differences were observed for the clinical results in the group of couples with an affected female versus the group with an affected male or versus a group of couples with different referral reasons. Specifically, both the percentage of mature oocytes out of the total oocytes retrieved and the percentage of fertilization were considerably lower in the group of DM1 females. Our findings would suggest the possibility of achieving less favourable PGD outcomes in women with DM1 in comparison with other pathologies, although the underlying mechanism remains unknown

Preimplantation Genetic Diagnosis for Myotonic Dystrophy Type 1 and Analysis of the Effect of the Disease on the Reproductive Outcome of the Affected Female Patients

Myotonic dystrophy type 1 (DM1) is the most common adult muscular dystrophy and presents an autosomal dominant inheritance. A reproductive option for the families affected is preimplantation genetic diagnosis (PGD). One limitation of this option is the nonoptimal response to ovarian stimulation of the women with DM1, although controversial results exist regarding this subject. In this study, we have analyzed the results of the PGD program applied to DM1 at our institution. A total of 35 couples have been included in our program since 2010, and 59 cycles have been performed. The percentage of transfers per cycle was 64.4% and the live birth rate per cycle was 18.6%. Interestingly, statistically significant differences were observed for the clinical results in the group of couples with an affected female versus the group with an affected male or versus a group of couples with different referral reasons. Specifically, both the percentage of mature oocytes out of the total oocytes retrieved and the percentage of fertilization were considerably lower in the group of DM1 females. Our findings would suggest the possibility of achieving less favourable PGD outcomes in women with DM1 in comparison with other pathologies, although the underlying mechanism remains unknown.“Fundacion Pública Andaluza Progreso y Salud" Consejería de Sanidad, Junta de Andalucí

Occurrence of Salmonella typhimurium resistance under sublethal/repeated exposure to cauliflower infusion and infection effects on Caernohabditis elegans host test organism

Resistant bacteria to antimicrobials are increasingly emerging in medical, food industry and livestock environments. The present research work assesses the capability of Salmonella enterica var Typhimurium to become adapted under the exposure to a natural cauliflower antimicrobial by-product infusion in consecutive repeated exposure cycles. Caenorhabditis elegans was proposed as in vivo host-test organism to compare possible changes in the virulent pattern of the different rounds treated S. enterica var Typhimurium and untreated bacterial cells. According to the obtained results, S. enterica var Typhimurium was able to generate resistance against a repeated exposure to cauliflower by-product infusion 5% (w/v), increasing the resistance with the number of exposed repetitions. Meanwhile, at the first exposure, cauliflower by-product infusion was effective in reducing S. enterica var Typhimurium (≈1 log10 cycle), and S. enterica var Typhimurium became resistant to this natural antimicrobial after the second and third treatment-round and was able to grow (≈1 log10 cycle). In spite of the increased resistance observed for repeatedly treated bacteria, the present study reveals no changes on C. elegans infection effects between resistant and untreated S. enterica var Typhimurium, according to phenotypic parameters evaluation (lifespan duration and egg-laying).Peer reviewe